Fig. 4: OsVIRK1 physically interacts with RSV CP.

a Co-IP assay showing that OsVIRK1 is associated with CP in N. benthamiana. OsVIRK1-GFP and CP or SP were transiently co-expressed in N. benthamiana leaves. The proteins were subjected to anti-GFP IP followed by western blot analysis with anti-GFP, anti-CP, and anti-SP antibodies. b GST pull-down assay showing that OsVIRK1 binds to CP, but not SP, in vitro. His-tagged OsVIRK1 (OsVIRK1-His), GST-tagged CP (CP-GST) and SP (SP-GST), and GST recombinant proteins were affinity-purified, and protein–protein interactions were examined by GST pull-down assay. c Co-IP assay showing that OsVIRK1 is associated with CP in RSV-infected rice. Total proteins were extracted from RSV-infected WT and OsVIRK1-FLAG-HA-OE rice plants, followed by anti-FLAG co-IP and subsequent western blot analysis with anti-HA and anti-CP antibodies. d, e BiFC assay showing that OsVIRK1 interacts with CP at the TGN/EE. The designated construct pairs were co-transfected into rice protoplasts and N. benthamiana leaves, and the CFP fluorescence signals were observed. Scale bars, 10 μm (d) or 50 μm (e). f Co-IP assay showing that both the N- and C-terminal regions of OsVIRK1 are associated with CP in N. benthamiana. GFP, OsVIRK1-GFP, OsVIRK1N-GFP, or OsVIRK1C-GFP was transiently co-expressed with CP-HA in N. benthamiana leaves. The proteins were then subjected to anti-GFP IP and subsequent western blot analysis using anti-GFP and anti-HA antibodies. The red stars mark the expected protein bands in the western blots. g, h MST analysis showing the binding affinity between OsVIRK1N-His or OsVIRK1C-His and CP-GST. All experiments were repeated two or three times with similar results.